Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.10.1 (ERK)
95,504 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have expressed in COS-1 cells mutants of neprilysin (neutral endopeptidase-24.11; NEP) in which the hydrophilic sequence S-Q-N-S was either substituted for V42-T-M-I or inserted after T38 in the signal peptide/membrane anchor (SA) domain. These mutations were introduced in full-length NEP (mutants NEP(H1) and NEP(H2), respectively) and a form of NEP lacking its cytosolic tail (mutants NEP delta cyto(H1) and NEP delta cyto(H2), respectively). Immunoblotting showed that NEP(H1) was membrane-bound while NEP delta cyto(H1), NEP(H2), and NEP delta cyto(H2) were secreted. Furthermore, carbonate treatment of isolated intracellular membranes suggested that cleavage of the SA domain was performed in the endoplasmic reticulum, presumably by signal peptidase. Sequencing of the secreted proteins indicated that cleavage of the SA domain mostly occurred at the carboxy side of Ala46 but also at the carboxy side of Ala41 in NEP(H2) and NEP delta cyto(H2). We conclude that the position of the S-Q-N-S sequence influences the accessibility of the cleavage site and, in the case of NEP(H1) and NEP(H2), the efficiency of cleavage of the SA domain.
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PMID:Insertion of hydrophilic amino acid residues in the signal peptide/membrane anchor domain of neprilysin (neutral endopeptidase-24.11) results in its cleavage: role of the position of insertion. 798 81

Bone marrow (BM) stromal cells express CD10 (cALLA), a surface antigen now known to be a neutral endopeptidase (NEP-24.11). The function of CD10 in BM stroma is unknown, although purified NEP-24.11 is known to degrade different substrates including interleukin 1 beta (IL-1 beta). We have therefore employed a CD10-positive BM stromal cell line (L2AK) which proliferates in response to IL-1 beta to test the hypothesis that degradation of this cytokine is one of the functions of stromal CD10. We first showed that [3H]thymidine incorporation by L2AK cells is enhanced by IL-1 beta in a clear dose-dependent manner. Addition of the CD10 inhibitor, phosphoramidon, together with IL-1 beta resulted in a left shift in the dose-response curve which corresponded to a 10-fold potentiation of the IL-1 beta effect. These results indicate that CD10 on bone marrow stromal cells can degrade IL-1 beta and therefore provide a local control of the effects of this, and possibly other, growth factor(s).
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PMID:A function of CD10 on bone marrow stroma. 799 15

Neutral endopeptidase (Endopeptidase 24.11; NEP; neprilysin), an integral membrane protein, and villin, a major microvillar cytoskeletal actin-binding protein, are both typically associated with brush border epithelia. In this study, cRNA probes were hybridized in situ to investigate the expression of NEP and villin genes in embryo and adult mouse enterocytes. During development, villin mRNAs were easily detected in the immature digestive tract well before establishment of the brush border. In 17-day-old embryos, a transient elevation of villin mRNA occurred just prior to a dramatic increase in microvilli length and density. NEP only appeared by day 17 as the embryonic gut began to become functional. It therefore appears that the onset of transcription of specialized cytoskeletal proteins from the brush border preceded that of intrinsic membrane-bound enzyme from microvilli. In the adult intestinal fold, both mRNAs were expressed along the whole length of the villus with maximal expression at its base. In contrast, both proteins were uniformly expressed along the whole crypt-villus axis. Quantitative analysis revealed an asymmetric intracellular distribution of both mRNAs that were differentially polarized in the apical cytoplasm of enterocytes.
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PMID:Comparative analysis of neutral endopeptidase (NEP) and villin gene expression during mouse embryogenesis and enterocyte maturation. 802 47

Inhibitors of the zinc protease neutral endopeptidase (NEP, EC 3.4.24.11) offer significant therapeutic interest as antihypertensives due to their ability to potentiate the biological action of the circulating natriuretic hormone ANF (atrial natriuretic factor). N-Phosphonomethyl dipeptides bearing a central (4-phenyl)phenylalanine residue have been designed to exert potent and selective NEP inhibition. In particular, (S)-3-[N-[2- [(phosphonomethyl)amino]-3-(4-biphenylyl)propionyl]amino]propionic acid (10a) (CGS 24592) displayed high inhibitory potency in vitro (IC50 = 1.9 +/- 0.1 nM) and a long plasma half-life in rats but lacked oral bioavailability. This drawback was overcome by using esterase-sensitive (acyloxy)alkyl phosphonates. More remarkable, several diaryl phosphonate derivatives of 10a also performed as effective prodrugs. Specifically, the structurally simple diphenyl phosphonate 18 (CGS 25462) induced potent inhibition of NEP ex vivo for at least 8 h after oral administration to rats (30 mg/kg). Its antihypertensive effect was demonstrated in DOCA-salt rats. At 30 mg/kg orally, 18 caused a significant reduction in mean arterial pressure measuring -35 +/- 7 mmHg at 5-h postdosing. The alpha-aminomethyl phosphonate 18 represents a new generation of selective NEP inhibitors that combine high potency, long duration of action, and oral bioavailability. Therefore, it holds promise as a novel therapeutic agent for the treatment of human hypertension and congestive heart failure.
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PMID:N-Phosphonomethyl dipeptides and their phosphonate prodrugs, a new generation of neutral endopeptidase (NEP, EC 3.4.24.11) inhibitors. 812 Aug 68

Alatriopril is a dual inhibitor of two cell surface metallopeptidases which play important roles in the regulation of arterial blood pressure and renal function: the angiotensin I converting enzyme (ACE) which catalyses transformation of angiotensin I to angiotensin II, and the neutral endopeptidase (NEP; EC 3.4.24.11; atriopeptidase), responsible for the degradation of the atrial natriuretic factor (ANF). The purpose of the present study was to evaluate the systemic and regional hemodynamic effects of alatrioprilat, the active part of alatriopril, in 6 anesthetized, closed-chest beagle dogs instrumented for the measurement of arterial pressure (aortic catheter), cardiac output (thermodilution), as well as femoral and renal artery flows (Doppler). Animal received alatrioprilat at the doses of 1 and 10 mg/kg (i.v. bolus). Hemodynamic parameters were measured at baseline, then 15, 30, 45 and 60 min after administration of each dose. In addition, plasma ANF and ACE activity were determined at baseline and 30 min after administration. At the dose of 1 mg/kg, alatrioprilat dit not induce marked hemodynamic effects, except a transient hypotension which appeared within the first 10 min after administration and lasted less than 10 min. Neither plasma ANF nor angiotensin converting enzyme activity were affected by this dose.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:[Systemic and regional hemodynamic effects of a new angiotensin converting enzyme and neutral endopeptidase mixed inhibitor, alatriopril, in the dog]. 812 43

A new metallo-endopeptidase which hydrolyzes atrium natriuretic factor (ANF) has been isolated from human neuroblastoma NB-OK-1 cells. In the present study we show that this metallo-endopeptidase is also present in several other human neuroblastoma cell lines, which include CHP 100, SH-SY5Y, SK-N-BE(2), BE(2)-C and BE(2)M-17. Additionally, we show that this endopeptidase activity is reduced to about 20% of the control during retinoic acid (RA)-induced neuronal differentiation in the RA-sensitive SK-N-BE(2) cells, but not in the RA-resistant BE(2)-M17 cells. This suggests that the inhibition is related to neuronal differentiation and not to a direct effect of 5 microM RA on the enzyme activity. This new enzyme is clearly distinct from neutral endopeptidase (NEP, EC 3.4.24.11) and angiotensin-converting enzyme (ACE,EC 3.4.15.1), since specific inhibitors for these endopeptidases (10 microM phosphoramidon and 1 mM captopril, respectively) had no effect on their activity. However, this enzyme was inhibited 100% by 10 mM o-phenanthroline showing an inhibitory spectrum similar to that of another novel metallo-endopeptidase recently isolated in our laboratory from Xenopus laevis skin secretion. Although the physiological function of this new enzyme in human neuroblastoma cells is not known at the present time, we suggest that it may participate in inactivation of neuropeptides such as atrium natriuretic factor (ANF), substance P, somatostatin-14 and bradykinin in vivo.
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PMID:Human neuroblastoma cells express a novel metallo-endopeptidase activity able to inactivate atrial natriuretic factor: inhibition during retinoic acid-induced differentiation. 813 18

Neutral endopeptidase 24.11 (NEP; "enkephalinase") may inactivate a number of centrally active neuropeptides including the enkephalins and substance P. In most areas of the central nervous system, the cell types which express NEP activity are not known. The hypoglossal nucleus (N.XII) was selected as a model system to characterize the cytochemical localization of NEP. The effect of hypoglossal nerve axotomy upon the distribution of NEP activity in the hypoglossal nucleus was compared to the effect upon cholinergic markers, the mu opiate receptor, and the enkephalins. By use of a fluorescence histochemical method, NEP was localized at all levels of N.XII to the soma and proximal processes of the majority of the apparent motor neurons in the nucleus. Fluorescent double-labeling studies revealed the presence of numerous enkephalinergic varicosities which localized to the neuropil surrounding NEP-stained motor neurons. To determine whether NEP was synthesized by these motor neurons, 18 rats received a unilateral transection of the hypoglossal nerve. A pronounced decrease in NEP staining in N.XII was observed on the operated side as early as 3 days following axotomy. This decrease persisted at all levels of the nucleus for about 5 weeks. By 7 weeks, the staining between the control and operated sides was indistinguishable. By contrast, there was no apparent change in the density or distribution of enkephalin-immunoreactive varicosities in five animals examined 6 to 32 days following axotomy. Radioligand binding of [3H]DAMGO to the mu-opiate receptor in N.XII was studied in 20 animals by quantitative autoradiography at 2, 6, and 11 days after axotomy. No significant changes in the level of radioligand binding to the mu-receptor were detected in response to axotomy. In contrast to the opiate system, the cholinergic enzymes choline acetyltransferase, acetylcholinesterase, and pseudocholinesterase showed a coordinate decrease in motor neuron-associated staining on the operated side of N.XII at 3, 6, and 11 days following axotomy which paralleled the decrease in NEP staining. By contrast, the lysosomal enzyme marker, acid phosphatase, showed a pronounced increase in staining on the operated side. The results of this study are consistent with the synthesis of NEP by cholinergic N.XII motor neurons and indicates that the enkephalins and NEP in N.XII are closely associated, but derive from separate neuronal populations. The widespread overlap in the distribution of NEP-stained motor neurons and enkephalinergic varicosities in N.XII provides additional anatomical support for a potential role for NEP in the inactivation of centrally active enkephalins.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Differential response of neutral endopeptidase 24.11 ("enkephalinase"), and cholinergic and opioidergic markers to hypoglossal axotomy. 820 Oct 16

Neutral endopeptidase (E.C.3.4.24.11, enkephalinase, NEP) is a potentially important enzyme capable of regulating the activity of neuropeptides released in the respiratory mucosa. In order to confirm the existence of NEP in the human respiratory mucosa, inferior nasal turbinate mucosae obtained at surgery and nasal secretions induced by topical provocations with methacholine, histamine, and allergen were analyzed for: (1) NEP activity (pmol product/min/ml) by enzymatic degradation of [3H]leu-enkephalin, (2) the presence of NEP-immunoreactive material by Western blot analysis, and (3) cellular localization of NEP distribution by immunohistochemistry. NEP activity in human nasal secretions obtained after normal saline challenge was 0.15 +/- 0.06 pmol/min/ml. Secretion increased to 0.86 +/- 0.26 pmol/min/ml after methacholine provocation and 1.69 +/- 0.74 pmol/min/ml after histamine provocation. The increase in NEP activity in methacholine-induced secretions was prevented by atropine (0.13 +/- 0.06 pmol/min/ml). After methacholine, histamine, and antigen nasal provocation, the kinetics of NEP appearance correlated more closely to the glandular marker, lactoferrin, than with the vascular markers albumin and IgG. In homogenates of nasal mucosa, the membrane fraction contained significantly more NEP on a per mg protein basis than did the soluble fraction (227.6 +/- 50.52 versus 9.61 +/- 3.18 pmol/min/mg protein, respectively, P < 0.01, n = 6). NEP in the membrane fraction was detected as a single band migrating at 97 kD on Western blots using antibodies specific for NEP and the common acute lymphoblastic leukemia antigen (CALLA). Immunoreactive NEP was localized to serous cells of the submucosal glands, epithelial cells, and endothelial and myoepithelial cells of small vessels. Staining for NEP in the serous cells was of the same intensity as that in epithelial cells. These results indicate that 97 kD NEP-immunoreactive material exists in discrete locations in the nasal mucosa, including the epithelium, serous cells of the submucosal glands, and vessel walls, and that NEP activity is detected as a minor component in nasal secretions enriched by glandular products. In addition to the modulating functions of NEP on neuropeptide-mediated activities on vessels and glands, it is possible that NEP in secretions plays a role in regulating mucosal responses to luminal neuropeptides or other as yet uncharacterized NEP substrates.
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PMID:Human nasal mucosal neutral endopeptidase (NEP): location, quantitation, and secretion. 821 97

The purpose of this study was to investigate whether angiotensin-converting enzyme (ACE; EC 3.4.15.1) and neutral endopeptidase (NEP; EC 3.4.24.11), two membrane-bound metalloenzymes that are widely distributed in the peripheral microcirculation and degrade kinins very effectively, modulate bradykinin-induced arteriolar dilation in vivo. Using intravital microscopy, we measured diameter of second-order arterioles in the hamster cheek pouch during suffusion of bradykinin (0.1-10.0 microM) before and after topical application of captopril (10.0 microM) and phosphoramidon (10.0 nM). We found that each inhibitor significantly potentiated bradykinin-induced increase in arteriolar diameter (P < 0.05). Suffusion of other proteinase inhibitors (excluding ACE and NEP inhibitors) had no significant effect on bradykinin-induced responses. Captopril and phosphoramidon did not potentiate isoproterenol (0.1 microM)-induced arteriolar dilation in the cheek pouch. Collectively, these data indicate that ACE and NEP each plays an important role in regulating bradykinin-induced vasorelaxation in the peripheral microcirculation in vivo.
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PMID:Peptidases modulate bradykinin-induced arteriolar dilation in the hamster cheek pouch. 830 27

Neutral endopeptidase 24.11 (NEP/CALLA/CD10), an enzyme expressed on early lymphoid progenitors, neutrophils, and various other cell types, inactivates many biologically active peptides, including the bacterial chemotactic peptide N-formylmethionyl-leucyl-phenylalanine (fMLP). Inhibition of CD10/NEP on the surface of human neutrophils (PMNs) in vitro inhibits migration toward this chemotaxin, suggesting that enzymatic inactivation by NEP regulates the neutrophil response to fMLP. Because PMNs in inflammatory sites are exposed to various cytokines, we evaluated the effects of selected cytokines on CD10/NEP activity in vitro. Of five cytokines tested--interleukin-1 (IL-1), IL-6, and IL-8, granulocyte colony-stimulating factor, and granulocyte-macrophage colony-stimulating factor (GM-CSF)--GM-CSF provided the most consistent increase in surface NEP activity. Low concentrations (10(-9)-10(-7) M) of GM-CSF increased NEP activity in a time- and concentration-dependent manner to more than 225% that of control (phosphate-buffered saline-treated) cells. Cytofluorometry of cells stained with a fluorescent antibody to CD10 indicated that GM-CSF increased expression of surface CD10/NEP antigen in a similar manner. The effect of GM-CSF on NEP activity was enhanced still further by simultaneous exposure to IL-1, suggesting that combinations of cytokines may direct and regulate the neutrophil response within an inflammatory site. Rapid upregulation of CD10/NEP underscores the importance of this enzyme for control of peptide mediators of inflammation.
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PMID:Up-regulation of neutral endopeptidase (CALLA) in human neutrophils by granulocyte-macrophage colony-stimulating factor. 831 51


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